Highly accurate detection of single molecules from biological sample and other samples can be widely used in medical diagnostics, pathology, toxicology, environmental sampling, chemical analysis, and many other areas, and nanoparticles and chemicals labeled with specific substances have been used in researches for metabolism, distribution and coupling of small amounts of synthetic substances and bio-molecules in biochemistry for last a few years. Typically, there are methods using radioactive isotopes, organic fluorescent materials and quantum dots which are inorganic materials.
3H, 14C, 32P, 35S and 125I, which are radioactive isotopes of 1H, 12C, 31P and 127I extensively found in the living body, are widely used as radioactive indicators in the method using radioactive isotopes. Radioactive isotopes have been used for a long time because of the similar chemical properties with non-radioactive isotope, which enables a random replacement, and relatively large emission energy, which enables the detection of small amounts. However, it is not easy to handle because of the harmful radiation and the radiation of some isotopes has short half-life instead of large emission energy, causing inconvenience in long-term storage or experiment.
Organic fluorescent dyes are widely used as alternatives to radioactive isotopes. Fluorescent dyes emit light with unique wavelength when activated by light with specific wavelength. Particularly, while radioactive material expresses the limitation in the detection, requiring long detection time with miniaturization of detection device, fluorescent dyes emit thousands of photons per molecules under appropriate conditions and theoretically enable the detection even at the level of a single-molecule. However, the fluorescent dyes have limitations in that the fluorescent dyes are connected by deformation of the part which relatively little affects the activity through structure activity relationship, incapable of direct substitution of the elements of the active ligand as radioactive isotopes. In addition, these fluorescent markers emit weaker intensity of fluorescence over time (photobleaching) and have a very narrow wavelength range of activation light and a wide wavelength range of emission light leading to the disadvantage of interference between different fluorophores. Also, the number of available fluorophores is extremely limited.
Also, semiconductor nano materials, quantum dots, is composed of CdSe, CdS, ZnS, ZnSe, etc. and emit lights of different colors depending on the size and type. Quantum dots, with wide active wavelengths and narrow emission wavelength compared to organic fluorescent dyes, have larger number of cases in which light of different colors are emitted than organic fluorescent dyes. In recent years, therefore, quantum dots have been used as a way to overcome the shortcoming of organic fluorescent dyes. However, they have disadvantages of high toxicity and difficulty of mass production. In addition, the number of available quantum dots, although theoretically variable, is highly restricted in practice.
To overcome such problems, Raman Spectrometry and/or Surface Plasmon Resonance have been recently used for labeling.
Among them, Surface Enhanced Raman Scattering (SERS) is the spectroscopy using the phenomenon that the intensity of Raman scattering increases rapidly by more than 106 to 108 times when the molecule is adsorbed on the roughened surface of metallic nanostructure of gold, silver, etc. When the light passes through a concrete medium, a certain amount of light deviates from an unique direction, which is known as Raman scattering. Since some of the scattered light is absorbed and excites an electron to the higher level of energy, the wavelength of Raman emission spectrum is different from that of stimulated light and represents the chemical composition and structural properties of light absorbing molecule in the sample. Therefore, Raman spectroscopy, combined with rapidly advancing current nanotechnology, can be developed into the highly sensitive technology to detect directly a single molecule and is largely expected to be used especially as crucial medical sensor. The Surface Enhanced Raman Scattering (SERS) is related to plasmon resonance phenomenon, and since wherein metal nanoparticles shows the pronounced optical resonance in response to the incident electromagnetic radiation by group coupling of metal conduction electrons in the metal, the nanoparticles of gold, silver, copper and certain other metals can be used essentially as a small antenna to improve focusing effects of electromagnetic radiation. Molecules located in the vicinity of these particles represent a much greater sensitivity for Raman spectroscopy analysis.
Therefore, the researches for early diagnosis of various disease-associated genes and proteins (biomarkers) using SERS sensors are actively carried out. Unlike the other analysis methods (infrared spectroscopy), Raman spectroscopy has several advantages. While infrared spectroscopy obtains a strong signal in the case of molecules with change in the molecular dipole moment, Raman spectroscopy can obtain a strong signal even in the case of non-polar molecule, resulting that almost all organic molecules have a unique Raman shift (cm−1). In addition, because it is not affected by water molecules interference, Raman spectroscopy is more suitable for the detection of biomolecules such as proteins, genes, etc. However, due to the low signal intensity, it did not reach a level of practical use despite long research period.
In the continuous researches since the discovery of Surface-Enhanced Raman Scattering, researches regarding the SERS enhancement phenomenon using a variety of nanostructures (nanoparticles, nanoshells, or nanolines) have been reported after the Surface Enhanced Raman Scattering (SERS) which is capable of detection of the single molecular level of signal in the disordered aggregate of nanoparticles with fluorescent molecules adsorbed, was reported (science 1997, 275(5303), 1102; Phys rev lett 1997, 78(9), 1667). Mirkin and his team recently successfully achieved high sensitivity DNA analysis using gold nanoparticles combined with DNA to use the SERS phenomenon with high sensitivity in the development of bio-sensors, with detection limit of 20 fM (2002, science, 297, 1536). However, there has been little progress in the preparation methods for single-molecule SERS active substrates based on salt induced aggregation of silver (Ag) nanoparticles with the Raman active molecule (eg, Rhodamine 6G) since the initial study. It was reported that in the heterogeneous coagulated colloid, only a fraction (less than 1%) has single molecule SERS activity (J Phys Chem B 2002, 106(2), 311). Although randomly inhomogeneous (roughed) surface provides a large amount of interesting and essential data associated with SERS, such a strategy is essentially reproducible due to significant changes in enhancement by small surface morphological changes. Recently, Fang et al. reported the quantitative measurements of distribution of enhanced regions in SERS. The densest areas (EF>109) were reported as 64 areas out of total 1,000,000 areas, which contribute to 24% of the total SERS intensity (Science, 2008, 321, 388). If the structure in which the SERS signal can be maximized with the reproducibility can be obtained, it can be a very reliable ultra-sensitive biomolecule analysis method, and can be useful for in vivo imaging techniques as well as in vitro diagnostics.
However, in the previous SERS detection methods for the various analytes, the substrate and/or colloidal metal particles, such as aggregated silver nanoparticles, coated on the supporter were typically used, sometimes yielding SERS detection with increased sensitivity by 106 to 108 times, without being able to detect single-molecule of small analytes such as nucleotides. However, despite the advantages of SERS, the mechanism of SERS phenomenon are not only not fully understood, the preparation and control of well-defined nanostructures are also difficult, as well as many unsolved problems exist in terms of reproducibility and reliability arising from the changes in enhancement efficiency depending on the wavelength of the light used to measure the spectrum, and the polarization direction remains an unsolved problem for the application of the SERS phenomenon including the development and commercialization of nanobiosensors. Researchers for precise control of the SERS phenomenon are required to solve these problems by means of understanding the optical properties of well-defined nanostructures.
Heresupon, L. Brus et al. (JACS. 2002) reported in the case of dimer of metal particles, that a hot spot (interstitial field), which is a very strong electromagnetic field, is formed between two or more nanoparticles, resulting in SERS signal enhancement and SERS enhancement by hot spot is predicted as 1012 times according to theoretical electromagnetic calculations.
Thus, the enhanced sensitivity of Raman detection is not evidently homogeneous within colloidal particle aggregate, but depends on the presence of hot spots. However, the characteristics of the physical structure and distance range from nanoparticles, where enhanced sensitivity is achieved, of hot spots, and spatial correlation between the analytes to enhance the sensitivity and aggregate of nanoparticles have not been presented. In addition, the aggregated nanoparticles are inherently unstable in solution, and give an adverse effect on the reproducibility of the detection of single-particle analyte.
As far as the amplification of optical signal is concerned, characteristic amplified signal (eg, Raman, fluorescence, scattering, etc) of molecules emitting the optical signal located in the gap can be detected by the amplification of electromagnetic signals at the junction area outside two or more nanostructures. However, if surface-enhanced Raman scattering (SERS) is to be obtained using these structures, quantification of the signal, reproducibility of the results, ease and simplicity of synthesis, cost, and stability of the probe still remain the problems. In other words, if two or more nanoparticles are combined by a nanogap, the amplified optical signal detection is detectable, but simplicity of material synthesis, stability, reproducibility of the signal and quantification cannot be secured.
Therefore, the nanostructure which is capable of strong amplification of the signal is a single nanoparticle with a nanogap inside and, even though it has not been reported until now, it is expected that stable signal can be formed by placing various signal substances in the intra-nanogap.
Meanwhile, although synthesis and assembly of various nanostructures for DNA have been studied in-depth, there have been very few researches on other roles of DNA. Hereupon, the present inventors prepared single nanoparticle which includes core and shell with a nanogap formed between core and shell using DNA, away from the concept to form a nanogap using more than two nanoparticles. For the nanoparticle herein, especially when modifying the surface of the core by the DNA, part of the space between the core and the shell is connected by the nanobridge, and the nanogap can be adjusted to be formed between the core and the shell, the number and locations of Raman-active molecules can be easily adjusted by adjusting the nucleotide sequence of DNA, the synthesis thereof is simple, very high signal amplification effect is shown due to plasomonic coupling by intra-nanogap, and the problem of signal reproducibility and quantification, which is the crucial prerequisite to commercialization, is known to be overcome due to high reproducibility to complete the present invention.
The present inventors also identified the possibility to form a nanogap without nanobridge between core and shell by forming organic molecules (polymer, as one example, polymer layer with layer-by-layer structure of poly-allyl amine, poly-L-lysine, which is positively charged polymer, and negatively charged poly-styrene-sulfonate) which can combine with the surface of gold nanoparticle followed by forming the additional metal shell.